Project Summary The kidney plays a critical role in regulating potassium homeostasis by adjusting secretion in the distal nephron to match daily dietary intake. Distal K+ secretion involves K+ efflux through apical ROMK K+ channels, a process that requires Na+ reabsorption via ENaC to provide the electrical driving force for K+ secretion. The activity of the Na+- Cl- cotransporter, NCC, impacts distal K+ secretion by altering Na+ delivery to ENaC. Thus, besides upregulating ROMK, high dietary K+ intake enhances distal K+ secretion by downregulating NCC. WNK1 and WNK4 are protein kinases in which gain-of-function mutations cause an autosomal-dominant hypertension and hyperkalemia syndrome, pseudohypoaldosteronism type II, which occurs at least in part by increasing Na+ reabsorption via NCC and decreasing K+ secretion via ROMK. How dietary K+ intake regulates NCC and how WNK1 and 4 activate NCC and inhibit ROMK remain unresolved. As a part of long-term goal to understand the mechanism of renal Na+ and K+ transport in health and disease, we propose the following studies. Aim-1 will test the hypothesis that WNK1 and WNK4 both activate NCC in vivo and that it occurs via the OSR1/SPAK kinase cascade. We will examine the role of WNK1 and 4 on NCC in vivo using kidney-specific conditional Wnk1-knockout mice (to circumvent embryonic lethality of global Wnk1-deleted mice) and global Wnk4-deleted mice, respectively. Double knockout mice will be studied to investigate whether WNK1 and 4 are additive or antagonistic. The role of OSR1/SPAK in mediating WNK1/4 regulation of NCC will be investigated based on the ability of expression of catalytically constitutive-active OSR1/SPAK to rescue loss-of-function of NCC in mice lacking Wnk1 and/or Wnk4. The activity and expression of NCC in kidney will be assessed by analyzing the increase of urinary Na+ excretion in response to hydrochlorothiazide and western blot analysis of the total and phosphorylated NCC. Aim-2 will examine the hypothesis that WNK kinase cascade mediates dietary K+ intake-induced regulation of NCC. Abundant in vitro and cell-based evidence implicate that dietary K+ intake regulates NCC by altering intracellular chloride concentration to affect WNK kinase activity. We will test the hypothesis in vivo using mice carry Wnk4-null alleles and kidney- specific conditional Wnk1-knockout mice. Mice will be fed a normal K+ or low K+ diet and studied for NCC activity. Aim-3 will test hypothesis that WNK kinases can directly inhibit renal K+ secretion independently of the effect via increasing NCC-mediated Na+ reabsorption using whole animal clearance studies and in vitro microperfusion of cortical collecting ducts isolated from wild-type mice versus mice whose WNKs are deleted but NCC activity is normalized by constitutive-active SPAK/OSR1. These studies using state-of-the-art animal models and physiological approaches will provide important in vivo information for our understanding of the mechanism of renal Na+ and K+ handling in physiological and diseased states.